高浓度细胞分裂素诱导棉花悬浮细胞程序性死亡

棉花悬浮细胞经高浓度细胞分裂素（2mg／L KT或4mg／L KT）处理后第4天大量死亡，培养物呈褐黑色；抽提总DNA，进行琼脂糖胶电泳，结果表明：2mg／L KT和4mg／L KT两处理中有明显的大小为140～180bp及其倍增量片段的DNA“梯”。进行PI和FDA染色显微观察可以看到：2mg／L KT处理中大部分细胞的细胞核较小，核质浓缩，呈棒形或弯月形；胞质浓缩，出现了不对称的质壁分离，且丧失了FDA染色活性。这些结果表明，由高浓度细胞分裂素处理引起的棉花悬浮细胞大规模死亡是一种细胞程序性死亡（PCD）。不同的生理状态（UCSA和BAE）和不同生长时期（指数增长期和静止期）的棉花悬浮细胞对高浓度细胞分裂素的诱导反应存在差异。     

生长素和细胞分裂素对平邑甜茶组培苗PAs和NO含量的影响

 以平邑甜茶继代组培苗为试材,研究了NAA、IBA、ZT和6-BA作用下组培苗的生长、内源多胺（PAs）和一氧化氮（NO）含量的变化。结果表明,在MS培养基中添加0.02 ~ 1.00 mg·L^-1的NAA或IBA后,外植体鲜样质量增加率和蛋白质含量以及多胺含量和一氧化氮含量均明显增加,其中浓度在0.10 ~ 0.50 mg·L^-1 时增加最高;同样浓度下,NAA处理的外植体鲜样质量增加率和腐胺含量高于IBA。在0.08 ~ 4.00 mg·L^-1范围内,随着ZT或6-BA浓度的升高,外植体鲜样质量增加率、可溶性蛋白含量、多胺含量和一氧化氮含量均逐渐升高;同样浓度下,6-BA处理的外植体鲜样质量增率和腐胺含量高于ZT。生长素和细胞分裂素促进组培苗生长与促进内源PAs和NO的生成相伴随,组培苗生长动态与内源多胺和一氧化氮含量的变化趋势一致。     

Shortening the generation cycle in faba bean (Vicia faba) by application of cytokinin and cold stress to assist speed breeding

The aim of this study was to reduce the length of the breeding cycle for faba bean by accelerating seed setting. We examined the mode and time of exogenous 6-benzylaminopurine (BAP) (cytokinin) application, and cold treatments and their combinations in two faba bean genotypes. Acropetal node number of pod and seed set and pollen viability were recorded during the experiments. Application of BAP improved pollen germination. The application of 10^–5 M BAP 4 days after flowering increased seed set at the lower nodes. Cold treatment (8/4°C day/night for 2 days) after the onset of flowering induced the formation of more pods and faster pod set compared to the non-cold treatment. The time to first seed was significantly reduced, and pollen viability was increased in plants exposed to cold treatment. Increased pollen viability also showed a significant positive correlation with seed setting. The combinations of 10^–5 BAP and cold treatment together had similar and independent effects. These results will accelerate plant breeding in faba bean by providing additional tools for reducing generation time.     

葡萄细胞分裂素响应调节因子VvRR2互作蛋白筛选与鉴定

【目的】从葡萄中克隆细胞分裂素响应调节因子VvRR2,获得VvRR2的互作蛋白,为阐明VvRR2在欧洲葡萄抗病反应中的作用机制提供依据。【方法】对葡萄接种白粉病菌,提取总RNA后反转录,利用实时荧光定量PCR检测VvRR2转录本对白粉病菌的响应;构建瞬时表达载体pBI221-VvRR2-GFP,转化拟南芥原生质体进行亚细胞定位分析;构建酵母表达载体pGBKT7-VvRR2,转化酵母菌株AH109,检测VvRR2的转录激活活性;构建酵母表达cDNA文库,以VvRR2为诱饵,通过Mating法筛选互作蛋白,对获得候选序列进行Blast分析;将候选蛋白VvTGA的全长序列克隆至pGADT7载体形成重组载体pGADT7-VvTGA,与重组诱饵载体pGBKT7-VvRR2共转化酵母,进行双杂交验证VvRR2与VvTGA的相互作用;将VvTGA的全长序列克隆至pSPYNE（R）173载体,形成重组载体pSPYNE-VvTGA,将VvRR2的全长序列克隆至pSPYCE（M）载体,形成重组载体pSPYCE-VvRR2,然后将两个重组载体共转化拟南芥原生质体,利用双分子荧光互补技术验证VvRR2与VvTGA的相互作用。【结果】葡萄接种白粉病菌后,细胞分裂素响应调节因子VvRR2呈现受白粉病菌诱导表达模式。VvRR2定位在拟南芥原生质体的细胞核,转录激活试验结果表明VvRR2在酵母体内具有转录激活活性。在含有60 mmol·L^-1的3-AT培养基上可以抑制VvRR2诱饵载体的自激活活性,VvRR2诱饵载体对宿主酵母菌没有毒性。以VvRR2为诱饵,初步筛选到287个单克隆,在高严谨条件下进一步筛选获得23个有效序列,Blast分析显示这些基因参与蛋白质合成与降解、细胞分裂素信号传导、光反应和生物钟节律、生长发育和逆境响应。酵母回复双杂交试验结果显示含有空载体（pGADT7或pGBKT7）酵母在四缺培养基（含3-AT）上不能生长,含有两种重组质粒的酵母在四缺培养基（含3-AT）上能够生长,并在含有X-α-Gal的四缺培养基上能够显色。双分子荧光互补试验结果显示共转化pSPYCE-VvRR2与pSPYNE（R）173、pSPYNE-VvTGA与pSPYCE（M）的原生质体没有黄色荧光,而共转化pSPYCE-VvRR2与pSPYNE-VvTGA的原生质体显示黄色荧光。VvTGA的表达类似于VvRR2,呈现受白粉病菌诱导表达模式。【结论】葡萄细胞分裂素响应调节因子VvRR2是一个受白粉病菌诱导表达的转录因子,能够与VvTGA相互作用,并且VvTGA受白粉病菌诱导表达。     

Effect of Synthetic Cytokinin Application on Pod Setting of Individual Florets within Raceme in Soybean

Abstract

The application of synthetic cytokinin (6-benzylaminopurine, BA) to racemes of soybean genotype IX93-100 at 7 days after anthesis (DAA) enhanced pod-set percentage of the florets at the 5th position and above (numbered from the base on rachis). The endogenous cytokinin (trans-zeatin riboside) content of individual florets was measured at the 1, 3, 5, 7th position every 3 days after anthesis. Cytokinin was detected only from the florets at 9 DAA, and the content was higher in the more proximal florets while it became negligible in the 7th floret. These results suggest that an increase in the amount of cytokinin in individual florets might enhance the pod setting of the florets positioned at the middle or distal part within the raceme.     

红花萱草的花茎花蕾组织快繁技术的研究

取红花萱草的花蕾、花茎外植体,在MS培养基上用不同浓度的细胞分裂素BA和生长素NAA进行培养。结果表明:花茎的分化率比花蕾高,而最适合的培养基配方是:MS+BA 0.5 mg/L+NAA 0.1 mg/L+蔗糖30 g/L+琼脂8 g/L。可诱导分化出愈伤组织,继代培养愈伤组织增埴的同时可获得健壮的再生植株。最适的生根培养基为:MS+NAA 0.3 mg/L+活性碳0.3 g/L+蔗糖30 g/L+琼脂8 g/L。     

Spontaneous induction of tetraploidy in hop using adventitious shoot regeneration method

In the process of breeding triploid hop cultivars, diploid varieties need to be raised to the tetraploid level. Existing methods are predominantly based on treatment of the apices with antimitotic substances, in vivo or in vitro , resulting in a reported maximum of 26% tetraploids and a large proportion (over 60%) of mixoploids. This study was set-up to test whether an adventitious shoot regeneration protocol can be used as an alternative. In the first part of the experiments, regeneration media and a choice of explants (internodes, petioles and leaf discs) were optimized using three hop cultivars with putatively low, medium or high adventitious shoot regeneration capacity. On an optimized induction medium supplemented with 6 mg/l N ⁶-(2-isopentenyl) adenine (2iP), a regeneration rate up to 17.7% was obtained from cultured internodes, even of the most recalcitrant 'Aurora', while the regeneration rate of the most responsive 'Tettnanger' was 56.9%. Flow cytometric analysis of ploidy level revealed a high frequency of tetraploid induction, the highest (58.6%) being in 'Savinjski golding'. In 616 regenerants of all three cultivars, 30.4% were found to be tetraploid, the others were diploid. Cytokinin content was found to be of minor importance in tetraploid induction. The high frequency of spontaneous tetraploid induction and, in particular, the complete absence of mixoploids makes this protocol an alternative to the currently established methods of chromosome doubling based on the use of antimitotic substances. The occurrence of a high proportion of tetraploid adventitious regenerants in hop is discussed in view of certain other biotechnological applications.     

Callus Induction of Aloe vera L var chinensis (Haw) Berg

Studies were given to the induction and growth of callus of Aloe vera L.var.chinensis(Haw.) Berg when the young stem was applied as explant.MS was taken as basic medium,added with various concentrations of auxins(IAA,IBA,NAA and 2,4-D),cytokinins(6-BA,KT and Zeatin) and the different combinations of these plant growth regulators.Results showed that the callus is barely induced by cytokinin alone,and the callus induction rates have significant difference when different concentrations of auxin and different combinations of auxin with cytokinin are used on the MS medium.In order to inhibit callus browning during culturing,various concentrations of vitamin C or active carbon are added into four combinations of auxin and cytokinin respectively,which can induce callus most effectively.The result showsd that vitamin C can alleviate callus browning more effectively than the active carbon.The most efficient medium for induction and growth of callus of Aloe vera L.var.chinensis(Haw.) Berg was MS+2.0 mg/L 6-BA+0.5 mg/L 2,4-D+10 mg/ L vitamin C,with which the callus induction rate can reach to 91.4% and callus grow vigorously.     

细胞分裂素促进白杨派树种插穗生根作用的研究

用细胞分裂素(Cyt)配合生长素(Aux)处理银白杨、毛白杨和山杨插穗的试验表明,增加Cyt有促进愈伤组织发育和不定根形成的作用。与Aux处理相比,Aux+Cxt处理可使愈伤组织生根时间从50d缩短至30d左右。在人工气候室条件下,山杨扦插生根率达85％。     

冬季修剪对苹果新根细胞分裂素含量的影响研究

本试验以盆载３年生新红星／海棠为试材，应用高效液相色谱分析方法，研究了冬季修剪对苹果新根内源细胞分裂素含量的影响。新根中玉米素核苷（ＺＲ）、玉米素（Ｚ）的含量，缓放植株高于短截植株；但ＺＲ／Ｚ的比值，缓放植株低于短截植株。